Methods for characterizing disulfide bonds

ABSTRACT

Compositions and methods for analyzing disulfide bonds are provided. An exemplary method includes preparing peptide standards having no disulfide bonds, scrambled disulfide bond peptide standards, and native disulfide bond peptide standards according to the sequence of the region of the protein drug product that includes the disulfide bond, digesting a sample of protein drug product into peptides, separating the protein drug product peptides, analyzing the protein drug product peptides and the peptide standards, identifying scrambled and native disulfide bond peptides by retention time, and quantifying the level of scrambled disulfide bond peptides.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims benefit of and priority to U.S. ProvisionalPatent Application No. 62/792,994 filed Jan. 16, 2019, incorporatedherein by reference in its entirety.

TECHNICAL FIELD OF THE INVENTION

The invention is generally related to systems and methods ofcharacterizing antibodies, in particular disulfide bonds.

REFERENCE TO SEQUENCE LISTING

The Sequence Listing submitted Jan. 16, 2020, as a text file named“064752_027US1_seq_listing”, and having a size of 1,086 bytes is herebyincorporated by reference pursuant to 37 C.F.R. § 1.52(e)(5).

BACKGROUND OF THE INVENTION

During the development of monoclonal antibodies (mAbs) from drugcandidate to marketed product, issues with stability, post-translationalmodifications, or other changes to the antibody can occur. Alterationsin antibody structure and function can cause problems such as poorshelf-life or even immunogenicity in the patient. It is thereforeimportant to properly characterize antibody structure and monitor itthroughout production. Antibody quality control and quality assuranceare critical to the purity and safety of mAb products.

Disulfide bonds are important for structural integrity, stability, andbiological functions of mAbs. Non-native disulfide bonds can causechanges in the structure and stability of mAbs. Binding affinity of mAbsto antigens can be affected by up to 50% if disulfide bonds areincomplete (Xiang, T., et al., Anal Chem, 81:8101-8108 (2009)). The lowdissociation energy of disulfide bonds and the high flexibility of thehinge region frequently lead to modifications and cleavages at the hingeregion (Moritz, B., and Stracke, J. O., Electrophoresis, 36:769-785(2017)). In addition, administration of non-native disulfide bondedstructures to humans has the potential to trigger unwanted immuneresponses. Analysis of disulfide bonds is therefore important forquality control assessment of mAbs. Current methods of analyzing mAbdisulfide bonds are time-consuming and labor intensive.

Therefore, it is an object of the invention to provide systems andmethods for characterizing monoclonal antibodies, in particulardisulfide bonds in monoclonal antibodies.

SUMMARY OF THE INVENTION

Compositions and methods for characterizing disulfide bonds areprovided. One embodiment provides a method for identifying scrambleddisulfide bonds in a protein drug product and includes the steps ofpreparing peptide standards having regions of the protein drug productcontaining one or more disulfide bonds. The peptide standards can bemade to contain each different kind of scrambled disulfide bond. Forexample one standard can include a crossed disulfide bond, and anotherstandard can include an intra-chain disulfide bond. FIG. 1A showsexemplary forms of disulfide bonds that can be present in the peptidestandard. In one embodiment, the peptide standard contains a normal orparallel disulfide bond. Each peptide standard has a different, knownliquid chromatography retention time compared to the other peptidestandards. The method includes digesting a sample of protein drugproduct into peptides, and analyzing a sample containing protein drugproduct peptides and the peptide standards using a liquid chromatographytandem mass spectrometry system (LC-MS² system). Peptides detected atthe retention times of the different standards are indicative to thepresence in the protein drug product of the type of disulfide bond inthe specific peptide standard. In one embodiment, the protein drugproduct is a monoclonal antibody. In other embodiments, the protein drugproduct is a recombinant protein, a fusion protein, or a combinationthereof.

The peptide standards can be prepared using conventional techniques. Forexample an oxidation reaction can be used to generate disulfide bonds inthe peptide standards. In one embodiment, the oxidation reaction isperformed using Cu²⁺.

Another embodiment provides a method of producing a protein drug productincluding the steps of producing the protein drug product in a cellculture and identifying scrambled disulfide bonds of the protein drugproduct using the method describe above. The method includes modifyingone or more cell culture, purification or excipient conditions to reducethe amount of crossed hinge disulfide bonds of the protein drug productto less than 1.0%. The one or more conditions can include cell cultureconditions such as temperature, pH, oxygen levels, reactive oxygenspecies, surfactants, or combinations thereof.

Another embodiment provides a pharmaceutical composition includingmonoclonal antibodies having less than 30% scrambled disulfide bonds

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a schematic illustration showing the oxidation of a reducedhinge peptide having the sequence THTCPPCPAPELLG (SEQ ID NO:1). FIG. 1Bis a chromatogram showing hinge peptides that were oxidized overnight,resulting in peptides with two crossed disulfide bonds and hingepeptides with two parallel disulfide bonds. The peptides have thesequence THTCPPCPAPELLG (SEQ ID NO:1). FIG. 1C is a chromatogram showingpeptides with one parallel (Peak 1 and 3) or crossed (Peak 2) disulfidebond. The X axis represents time (minutes) and the Y axis representsabundance.

FIGS. 2A-2B are chromatograms showing disulfide bond formation inpeptides that were incubated with Cu²⁺ overnight. The peptides wereeither ˜0.1 μg/ml (FIG. 2A) or ˜8 μg/ml (FIG. 2B). The peptides have thesequence THTCPPCPAPELLG (SEQ ID NO:1). The X axis represents time(minutes) and the Y axis represents relative abundance.

FIGS. 3A-3H are chromatograms showing the results of N-terminal analysisof the parallel hinge peptide standard. The X axes represent time(minutes) and the Y axes represent mV.

FIG. 31 is a schematic illustration of the various peptides that can bedetected during the cycles of N-terminal analysis. The peptide sequencesare as follows: THTCPPCPAPELLG (SEQ ID NO:1), C-PTH (SEQ ID NO:2), andPPCPAPELLG (SEQ ID NO:3).

FIGS. 4A-4H are chromatograms showing the results of N-terminal analysisof the crossed hinge peptide standard. The X axes represent time(minutes) and the Y axes represent mV.

FIG. 41 is a schematic illustration of the various peptides that can bedetected during the various cycles of N-terminal analysis. The peptidesequences are as follows:

(SEQ ID NO: 1) THTCPPCPAPELLG,  (SEQ ID NO: 2) C-PTH,   and(SEQ ID NO: 3) PPCPAPELLG .

FIGS. 5A and 5B are chromatograms showing results from LC-MS analysis ofthe remaining peptides after four cycles of Edman degradation. FIG. 5Ashows peptides with crossed disulfide bonds and FIG. 5B shows peptideswith parallel disulfide bonds. FIG. 5C-5F are chromatograms of theindividual peptides from FIG. 5A. The peptide sequences are as follows:C-PTH (SEQ ID NO:2) and PPCPAPELLG (SEQ ID NO:3).

FIG. 6A is a chromatogram of a parallel hinge peptide standard for IgG1mAb1. FIG. 6B is a chromatogram of a crossed hinge peptide standard forIgG1 mAb. FIG. 6C is a chromatogram of IgG1 mAb1 peptides. The X axisrepresent time (minutes) and the Y axis represents relative abundance.The peptides have the sequence THTCPPCPAPELLG (SEQ ID NO:1).

FIG. 7A is a chromatogram of a parallel hinge peptide standard for IgG4mAb1. FIG. 7B is a chromatogram of a crossed hinge peptide standard forIgG4 mAb1. FIG. 7C is a chromatogram of IgG4 mAb1 peptides. The X axisrepresent time (minutes) and the Y axis represents relative abundance.The peptides have the sequence YGPPCPPCPAPEFLG (SEQ ID NO:4).

DETAILED DESCRIPTION OF THE INVENTION I. Definitions

It should be appreciated that this disclosure is not limited to thecompositions and methods described herein as well as the experimentalconditions described, as such may vary. It is also to be understood thatthe terminology used herein is for the purpose of describing certainembodiments only, and is not intended to be limiting, since the scope ofthe present disclosure will be limited only by the appended claims.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this disclosure belongs. Although any compositions,methods and materials similar or equivalent to those described hereincan be used in the practice or testing of the present invention. Allpublications mentioned are incorporated herein by reference in theirentirety.

The use of the terms “a,” “an,” “the,” and similar referents in thecontext of describing the presently claimed invention (especially in thecontext of the claims) are to be construed to cover both the singularand the plural, unless otherwise indicated herein or clearlycontradicted by context.

Recitation of ranges of values herein are merely intended to serve as ashorthand method of referring individually to each separate valuefalling within the range, unless otherwise indicated herein, and eachseparate value is incorporated into the specification as if it wereindividually recited herein.

Use of the term “about” is intended to describe values either above orbelow the stated value in a range of approx. +/−10%; in otherembodiments the values may range in value either above or below thestated value in a range of approx. +/−5%; in other embodiments thevalues may range in value either above or below the stated value in arange of approx. +/−2%; in other embodiments the values may range invalue either above or below the stated value in a range of approx.+/−1%. The preceding ranges are intended to be made clear by context,and no further limitation is implied. All methods described herein canbe performed in any suitable order unless otherwise indicated herein orotherwise clearly contradicted by context. The use of any and allexamples, or exemplary language (e.g., “such as”) provided herein, isintended merely to better illuminate the invention and does not pose alimitation on the scope of the invention unless otherwise claimed. Nolanguage in the specification should be construed as indicating anynon-claimed element as essential to the practice of the invention.

“Protein” refers to a molecule comprising two or more amino acidresidues joined to each other by a peptide bond. Protein includespolypeptides and peptides and may also include modifications such asglycosylation, lipid attachment, sulfation, gamma-carboxylation ofglutamic acid residues, alkylation, hydroxylation and ADP-ribosylation.Proteins can be of scientific or commercial interest, includingprotein-based drugs, and proteins include, among other things, enzymes,ligands, receptors, antibodies and chimeric or fusion proteins. Proteinsare produced by various types of recombinant cells using well-known cellculture methods, and are generally introduced into the cell by geneticengineering techniques (e.g., such as a sequence encoding a chimericprotein, or a codon-optimized sequence, an intronless sequence, etc.)where it may reside as an episome or be integrated into the genome ofthe cell.

“Antibody” refers to an immunoglobulin molecule consisting of fourpolypeptide chains, two heavy (H) chains and two light (L) chainsinter-connected by disulfide bonds. Each heavy chain has a heavy chainvariable region (HCVR or VH) and a heavy chain constant region. Theheavy chain constant region contains three domains, CH1, CH2 and CH3.Each light chain has a light chain variable region and a light chainconstant region. The light chain constant region consists of one domain(CL). The VH and VL regions can be further subdivided into regions ofhypervariability, termed complementarity determining regions (CDR),interspersed with regions that are more conserved, termed frameworkregions (FR). Each VH and VL is composed of three CDRs and four FRs,arranged from amino-terminus to carboxy-terminus in the following order:FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The term “antibody” includesreference to both glycosylated and non-glycosylated immunoglobulins ofany isotype or subclass. The term “antibody” includes antibody moleculesprepared, expressed, created or isolated by recombinant means, such asantibodies isolated from a host cell transfected to express theantibody. The term antibody also includes bispecific antibody, whichincludes a heterotetrameric immunoglobulin that can bind to more thanone different epitope. Bispecific antibodies are generally described inU.S. Pat. No. 8,586,713, which is incorporated by reference into thisapplication.

“Hinge region” refers to the flexible amino acid stretch in the centralpart of the heavy chains of the IgG and IgA immunoglobulin classes,which links these 2 chains by disulfide bonds. In IgG immunoglobulinsthe hinge region is located between the CH1 and CH3 constant domains.The hinge region affords flexibility to the antibody, and allows easierbinding to the antigen.

“Fc fusion proteins” comprise part or all of two or more proteins, oneof which is an Fc portion of an immunoglobulin molecule, which are nototherwise found together in nature. Preparation of fusion proteinscomprising certain heterologous polypeptides fused to various portionsof antibody-derived polypeptides (including the Fc domain) has beendescribed, e.g., by Rath, T., et al., Crit Rev Biotech, 35(2): 235-254(2015), Levin, D., et al., Trends Biotechnol, 33(1): 27-34 (2015))“Receptor Fc fusion proteins” comprise one or more extracellulardomain(s) of a receptor coupled to an Fc moiety, which in someembodiments comprises a hinge region followed by a CH2 and CH3 domain ofan immunoglobulin. In some embodiments, the Fc-fusion protein comprisestwo or more distinct receptor chains that bind to one or more ligand(s).For example, an Fc-fusion protein is a trap, such as for example an IL-1trap or VEGF trap.

The term “disulfide bond” refers to the linkage formed by the oxidationof two SH groups, each attached to a cysteine. Disulfide bonds play animportant role in the folding and stability of many proteins. IgGsinclude two heavy chains (HC) and two light chains (LC) covalentlylinked by a total of 16 inter- or intra-molecular disulfide bonds. IgGmAbs contain 32 cysteine residues, 5 cysteine residues on each LC and 11cysteine residues on each HC. Each LC contains one variable domain andone constant domain with a disulfide bond connection. The 5^(th)cysteine on the LC is linked to either the 3^(rd) or 5^(th) cysteine ofthe HC to form an interchain disulfide bond. The heavy chains include anN-terminal variable domain (VH) and three constant domains (CH1, CH2,and CH3) with a hinge region between CH1 and CH2 (Vidarsson, G., et al.,Front Immunol, 5:520 (2014)). The 6th and 7th cysteine on each HC arebonded forming the hinge region. The hinge region of an immunoglobulinhelps form the Y-shaped structure of the immunoglobulin molecule. The Yshape makes possible the flexibility of the immunoglobulin moleculesrequired in antigen binding.

“Intra-chain disulfide bond” refers to bonds that are formed between twocysteines within the same protein chain.

“Inter-chain disulfide bond” refers to bonds that are formed between twocysteines of individual chains of the same protein or between twocysteines of distinct proteins.

“Scrambled disulfide bond” refers to a disulfide bond in which acysteine bonds to a cysteine to which it does not normally bond. Forexample, cysteine X binds to cysteine Z instead of cysteine Y. Exemplaryscrambled disulfide bonds include but not limited to crossed andintra-chain disulfide bonds.

As used herein, the term “crossed-hinge” refers to an antibody hingeregion in which the disulfide bonds within the hinge region of theantibody are in a crossed instead of parallel formation as seen in thebottom right of FIG. 1A.

The term “LC-MS” refers to liquid chromatography-mass spectrometry whichis an analytical chemistry technique that combines the physicalseparation capabilities of liquid chromatography (or HPLC) with the massanalysis capabilities of mass spectrometry (MS).

II. Methods of Characterizing Disulfide Bonds

Disulfide bonds are critical for IgG tertiary structure, stability, andbiological function. Cysteine residues are involved in disulfide bonds.Each subclass of human IgG molecules has a well-defined homogenousdisulfide structure; however, there are many reported cases in whichdisulfide bond heterogeneity exists. Any two cysteines in closeproximity will form a covalent bond, even cysteines that do notnaturally pair together. The formation of disulfide bonds betweennon-naturally paired cysteines is called scrambling or aggregation.Disclosed herein are different methods for identifying disulfide bonds.Also disclosed herein are methods for producing protein drug productswith less than 30% scrambled disulfide bonds

A. Characterizing Disulfide Bonds

Analysis of disulfide bonds is important for quality control assessmentof mAbs. In one embodiment, the disulfide bonds are in the hinge region.Traditional methods for mAb hinge region disulfide bond pattern analysisinvolves proteolysis, fractionation and Edman degradation analysis,which is time-consuming and labor-intensive. In addition, traditionalmethods such as MS²-based techniques fail to distinguish between crossedand parallel hinge peptides. Identifying scrambled disulfide bonds isdifficult because of the very low number of scrambled disulfide bondsthat occur. Antibodies with scrambled disulfide bonds in the hingeregion can be less stable and have a potential for inducingimmunogenicity if administered to a subject. Disclosed herein arecompositions and methods of use thereof for characterizing disulfidebonds in proteins, for example monoclonal antibodies. Peptide standardswith native and scrambled disulfide bond patterns are provided herein.These peptide standards can be used in mass spectrometry analysis tofocus the analysis on peptides that elute with the peptide standards.Methods of applying the disclosed peptide standards to hinge regiondisulfide bond characterization are also provided.

1. Peptide Standards for mAb Disulfide Bond Pattern Analysis

In one embodiment the peptide standard is formed by two peptidescovalently bound together by one or more disulfide bonds. The scrambleddisulfide bonds occur when a disulfide bond forms between two aminoacids that are not directly opposite of each other. FIG. 7A shows thenatural parallel disulfide bond. FIG. 7B shows an exemplary crosseddisulfide bond also referred to as a scrambled disulfide bond. FIG. 1Ashows parallel, crossed and intra-chain disulfide bonds. In oneembodiment, the peptide standards can be used to identify the presenceof parallel, crossed, or intra-chain disulfide bonds in a proteinsample, for example scrambled disulfide bonds or native disulfide bondsin an antibody, for example a monoclonal antibody. In anotherembodiment, the peptide standards can detect intra-chain disulfide bondsin a protein or peptide. Further details for making and using thedisclosed disulfide bond peptides are provided below.

i. Synthesis

One embodiment provides a method for synthesizing disulfide bond peptidestandards. Peptide standards can be synthesized using techniques knownin the art, including but not limited to liquid phase synthesis, solidphase peptide synthesis, and recombinant technology (Stawikowski, M.,and Fields, G. B., Current Protoc Protein Sci, Chapter: Unit 18.1(2002)).

The peptide standards can include fragments of the protein containingthe disulfide bonds to be analyzed. The protein can be fragmented orsections of the protein containing the disulfide bonds to be analyzedcan be synthesized and used to produce disulfide bond peptide standards.In some embodiments, the peptide standard sequence has 100% sequenceidentity to the region of the protein or protein drug product ofinterest that includes the disulfide bond. In other embodiments, thepeptide standard sequence has at least 90% sequence identity to theregion of the protein or protein drug product of interest that includesthe disulfide bond. The peptide standard sequence can have 75%, 80%,85%, 90%, 95%, 97%, 98%, or 99% sequence identity to the region of theprotein or protein drug product of interest that includes the disulfidebond. In other embodiments, the sequence of the peptide standard onlyrepresents a portion of the region that includes the disulfide bond. Thepeptide standards are typically 5 to 20 amino acids in length.

The formation of disulfide bonds in the peptide standards can be inducedthrough oxidation of cysteine residues in the peptide standard. Methodsof forming disulfide bonds at cysteine include but are not limited toair oxidation, chemical oxidation, and exposing the peptide to copper(Cu²⁺) or zinc. Air oxidation occurs by mixing thiol and cysteinecontaining peptides in buffer open to the air. In another embodiment,the formation of disulfides on a peptide can be accomplished bydisulfide exchange, for example by using 5,5-dithiobis(2-nitrobenzoate)(DTNB or Ellman's Reagent). In one embodiment, other common chemicalsfor inducing oxidation of cysteine residues are activated reagents,including but not limited to iodine, sulfenyl halides, iodoacetamides,maleimides, benzylic halides and bromomethylketones. In anotherembodiment, disulfide bonds can be formed by exposing the peptide tocopper or zinc. This can be achieved by using an inert platinumelectrode or a sacrificial electrode (copper or zinc) or by generatingmetallic ions in electrospray ionization mass spectrometry (ESI-MS). Inone embodiment, the molar ratio of peptide:Cu²⁺ needed to induce theformation of disulfide bonds is 5:1 Higher peptide concentration canpreferentially induce the formation of hinge dimers over the formationof intra-chain disulfide bonds.

The peptide standards can be exposed to Cu²⁺ to induce single parallelor crossed disulfide bonds. In one embodiment, the peptide standards areoxidized for about 1 hour to about 6 hours. In a preferred embodiment,the peptide standards are oxidized for 2 hours. In another embodiment,the peptide standards can be oxidized for up to 24 hours in order toinduce two or more parallel or crossed disulfide bonds.

ii. Authentication

In one embodiment, the characteristics of the synthesized peptidestandards are determined. The characteristics include the retention timeand m/z of each peptide standard. The synthesized peptide standards canbe separated or fractionated using various chromatography methods.Peptide standards containing parallel disulfide bonds aredistinguishable from peptide standards containing crossed or intra-chaindisulfide bonds.

In one embodiment, N-terminal sequence analysis can be used to confirmthe identity of the peptide standards. N-terminal sequence analysisinvolves a series of chemical reactions that derivatize and remove oneamino acid at a time from the N-terminus of purified peptides or intactproteins. N-terminal analysis can detect disulfide bonds because thereaction to remove one amino acid at a time from the N-terminus does notdisrupt the bonds between the cysteine residues in the disulfide bond.

In another embodiment, Edman degradation can be utilized to sequence thedisulfide bond peptide standards. Edman degradation is similar toN-terminal analysis in that it detects the sequence of a protein orpeptide in order by removing one amino acid at a time from theN-terminus of the protein or peptide. However, the first round of Edmandegradation is often contaminated by impurities and therefore does notgive an accurate determination of the N-terminal amino acid. Edmandegradation can detect disulfide bonds because the reaction to removeone amino acid at a time from the N-terminus does not disrupt the bondsbetween the cysteine residues in the disulfide bond. In one embodiment,N-terminal analysis can be combined with Edman degradation to give acomplete, ordered sequence of the synthesized disulfide bond peptidestandards.

Other methods of sequencing peptides are considered. These include butare not limited to C-terminal analysis and mass spectrometry.

2. Methods for Characterizing Disulfide Bonds in the Hinge Region

One embodiment provides methods for identifying and characterizingdisulfide bonds in a protein drug product. In another embodiment, themethods identify and characterize disulfide bonds specifically in thehinge region of an antibody. In one embodiment, the antibody is an IgGantibody. An exemplary method includes preparing scrambled disulfidebond peptide standards and native disulfide bond peptide standardsaccording to the sequence of the protein drug product, cleaving a sampleof protein drug product into peptides, analyzing the peptide standardsand the protein drug product peptides, identifying scrambled and nativedisulfide bonds in peptides by comparing retention time, and quantifyingthe level of scrambled disulfide bond peptides. Detecting peptideshaving the same retention time or m/z as the peptide standard indicatesthat the type of disulfide bond in the peptide standard is present inthe protein drug product.

i. Protein Sample Preparation

The protein or protein drug product of interest can be obtained forexample from a bioreactor containing cells engineered to producemonoclonal antibodies.

In one embodiment, the protein or protein drug product of interest isdigested into peptides. Methods of digesting proteins are known in theart. Proteins can be digested by enzymatic digestion with proteolyticenzymes or by non-enzymatic digestion with chemicals. Exemplaryproteolytic enzymes for digesting proteins include but are not limitedto trypsin, pepsin, chymotrypsin, thermolysin, papain, pronase, Arg-C,Asp-N, Glu-C, Lys-C, and Lys-N. Combinations of proteolytic enzymes canbe used to ensure complete digestion. Exemplary chemicals for digestingproteins include but are not limited to formic acid, hydrochloric acid,acetic acid, cyanogen bromide, 2-nitro-5-thiocyanobenzoate, andhydroxyalamine.

In one embodiment, the protein drug product can be subjected to doubledigesting. In this embodiment, the first digestion can be performedusing a broad-specificity protease, such as but not limited toproteinase K, thermolysin, substilisin, papain, chymotrypsin, orelastase. The second digestion can be performed using trypsin. In oneembodiment, FabRICATOR® enzyme is used to digest the protein or proteindrug product. FabRICATOR® enzyme digests antibodies at a specific sitebelow the hinge therefore generating F(ab′)2 and Fc/2 fragments.FabRICATOR® digestion can be combined with tryptic digestion.

ii. Hinge Region Disulfide Bond Pattern Analysis

The digested peptide mixture from the protein or protein drug productcan be analyzed by liquid chromatography-mass spectrometry (LC-MS orLC-MS²) to determine the mass of the digested peptides. In oneembodiment, the digested peptide mixture is separated by liquidchromatography, for example size-exclusion chromatography.

The peptide mixture can then be analyzed using mass spectrometry.Methods of performing mass spectrometry are known in the art. See forexample (Aeberssold, M., and Mann, M., Nature, 422:198-207 (2003))Commonly utilized types of mass spectrometry include but are not limitedto tandem mass spectrometry (MS/MS), electrospray ionization massspectrometry, liquid chromatography-mass spectrometry (LC-MS), andMatrix-assisted laser desorption/ionization (MALDI). In anotherembodiment, selected reaction monitoring (SRM) is performed on thepeptide mixture. In SRM, an ion of a particular mass is selected in thefirst stage of a tandem mass spectrometer and an ion product offragmentation of the precursor ion is selected in the second massspectrometer for detection.

In one embodiment, the hinge peptide standards are also analyzed. Thestandards are used to characterize the hinge region of the protein drugproduct of interest. In one embodiment, the retention time of the knownhinge peptide standards are compared to the retention time of thepeptide mixture from the protein drug product of interest. Detectingpeptides having the same retention time or m/z as the peptide standardindicates that the type of disulfide bond in the peptide standard ispresent in the protein drug product.

B. Proteins of Interest

In one embodiment the protein of interest is a protein drug product oris a protein of interest suitable for expression in prokaryotic oreukaryotic cells. For example, the protein can be an antibody orantigen-binding fragment thereof, a chimeric antibody or antigen-bindingfragment thereof, an ScFv or fragment thereof, an Fc-fusion protein orfragment thereof, a growth factor or a fragment thereof, a cytokine or afragment thereof, or an extracellular domain of a cell surface receptoror a fragment thereof. Proteins in the complexes may be simplepolypeptides consisting of a single subunit, or complex multisubunitproteins comprising two or more subunits. The protein of interest may bea biopharmaceutical product, food additive or preservative, or anyprotein product subject to purification and quality standards

In some embodiments, the protein of interest is an antibody, a humanantibody, a humanized antibody, a chimeric antibody, a monoclonalantibody, a multispecific antibody, a bispecific antibody, an antigenbinding antibody fragment, a single chain antibody, a diabody, triabodyor tetrabody, a dual-specific, tetravalent immunoglobulin G-likemolecule, termed dual variable domain immunoglobulin (DVD-IG), an IgDantibody, an IgE antibody, an IgM antibody, an IgG antibody, an IgG1antibody, an IgG2 antibody, an IgG3 antibody, or an IgG4 antibody. Inone embodiment, the antibody is an IgG1 antibody. In one embodiment, theantibody is an IgG2 antibody. In one embodiment, the antibody is an IgG4antibody. In another embodiment, the antibody comprises a chimerichinge. In still other embodiments, the antibody comprises a chimeric Fc.In one embodiment, the antibody is a chimeric IgG2/IgG4 antibody. In oneembodiment, the antibody is a chimeric IgG2/IgG1 antibody. In oneembodiment, the antibody is a chimeric IgG2/IgG1/IgG4 antibody.

In some embodiments, the antibody is selected from the group consistingof an anti-Programmed Cell Death 1 antibody (e.g., an anti-PD1 antibodyas described in U.S. Pat. Appln. Pub. No. US2015/0203579A1), ananti-Programmed Cell Death Ligand-1 (e.g., an anti-PD-L1 antibody asdescribed in in U.S. Pat. Appln. Pub. No. US2015/0203580A1), ananti-D114 antibody, an anti-Angiopoetin-2 antibody (e.g., an anti-ANG2antibody as described in U.S. Pat. No. 9,402,898), ananti-Angiopoetin-Like 3 antibody (e.g., an anti-AngPt13 antibody asdescribed in U.S. Pat. No. 9,018,356), an anti-platelet derived growthfactor receptor antibody (e.g., an anti-PDGFR antibody as described inU.S. Pat. No. 9,265,827), an anti-Erb3 antibody, an anti-ProlactinReceptor antibody (e.g., anti-PRLR antibody as described in U.S. Pat.No. 9,302,015), an anti-Complement 5 antibody (e.g., an anti-05 antibodyas described in U.S. Pat. Appln. Pub. No US2015/0313194A1), an anti-TNFantibody, an anti-epidermal growth factor receptor antibody (e.g., ananti-EGFR antibody as described in U.S. Pat. No. 9,132,192 or ananti-EGFRvIII antibody as described in U.S. Pat. Appln. Pub. No.US2015/0259423A1), an anti-Proprotein Convertase Subtilisin Kexin-9antibody (e.g., an anti-PCSK9 antibody as described in U.S. Pat. No.8,062,640 or U.S. Pat. No. 9,540,449), an Anti-Growth andDifferentiation Factor-8 antibody (e.g. an anti-GDF8 antibody, alsoknown as anti-myostatin antibody, as described in U.S. Pat. Nos.8,871,209 or 9,260,515), an anti-Glucagon Receptor (e.g. anti-GCGRantibody as described in U.S. Pat. Appln. Pub. Nos. US2015/0337045A1 orUS2016/0075778A1), an anti-VEGF antibody, an anti-IL1R antibody, aninterleukin 4 receptor antibody (e.g., an anti-IL4R antibody asdescribed in U.S. Pat. Appln. Pub. No. US2014/0271681A1 or U.S. Pat.Nos. 8,735,095 or 8,945,559), an anti-interleukin 6 receptor antibody(e.g., an anti-IL6R antibody as described in U.S. Pat. Nos. 7,582,298,8,043,617 or 9,173,880), an anti-IL1 antibody, an anti-IL2 antibody, ananti-IL3 antibody, an anti-IL4 antibody, an anti-IL5 antibody, ananti-IL6 antibody, an anti-IL7 antibody, an anti-interleukin 33 (e.g.,anti-IL33 antibody as described in U.S. Pat. Nos. 9,453,072 or9,637,535), an anti-Respiratory syncytial virus antibody (e.g., anti-RSVantibody as described in U.S. Pat. Appln. Pub. No. 9,447,173), ananti-Cluster of differentiation 3 (e.g., an anti-CD3 antibody, asdescribed in U.S. Pat. Nos. 9,447,173 and 9,447,173, and in U.S.Application No. 62/222,605), an anti-Cluster of differentiation 20(e.g., an anti-CD20 antibody as described in U.S. Pat. No. 9,657,102 andUS20150266966A1, and in U.S. Pat. No. 7,879,984), an anti-CD19 antibody,an anti-CD28 antibody, an anti-Cluster of Differentiation-48 (e.g.anti-CD48 antibody as described in U.S. Pat. No. 9,228,014), an anti-Feld1 antibody (e.g. as described in U.S. Pat. No. 9,079,948), ananti-Middle East Respiratory Syndrome virus (e.g. an anti-MERS antibodyas described in U.S. Pat. Appln. Pub. No. US2015/0337029A1), ananti-Ebola virus antibody (e.g. as described in U.S. Pat. Appln. Pub.No. US2016/0215040), an anti-Zika virus antibody, an anti-LymphocyteActivation Gene 3 antibody (e.g. an anti-LAG3 antibody, or an anti-CD223antibody), an anti-Nerve Growth Factor antibody (e.g. an anti-NGFantibody as described in U.S. Pat. Appln. Pub. No. US2016/0017029 andU.S. Pat. Nos. 8,309,088 and 9,353,176) and an anti-Protein Y antibody.In some embodiments, the bispecific antibody is selected from the groupconsisting of an anti-CD3 x anti-CD20 bispecific antibody (as describedin U.S. Pat. Appln. Pub. Nos. US2014/0088295A1 and US20150266966A1), ananti-CD3 x anti-Mucin 16 bispecific antibody (e.g., an anti-CD3 xanti-Muc16 bispecific antibody), and an anti-CD3 xanti-Prostate-specific membrane antigen bispecific antibody (e.g., ananti-CD3 x anti-PSMA bispecific antibody). In some embodiments, theprotein of interest is selected from the group consisting of abciximab,adalimumab, adalimumab-atto, ado-trastuzumab, alemtuzumab, alirocumab,atezolizumab, avelumab, basiliximab, belimumab, benralizumab,bevacizumab, bezlotoxumab, blinatumomab, brentuximab vedotin,brodalumab, canakinumab, capromab pendetide, certolizumab pegol,cemiplimab, cetuximab, denosumab, dinutuximab, dupilumab, durvalumab,eculizumab, elotuzumab, emicizumab-kxwh, emtansinealirocumab,evinacumab, evolocumab, fasinumab, golimumab, guselkumab, ibritumomabtiuxetan, idarucizumab, infliximab, infliximab-abda, infliximab-dyyb,ipilimumab, ixekizumab, mepolizumab, necitumumab, nesvacumab, nivolumab,obiltoxaximab, obinutuzumab, ocrelizumab, ofatumumab, olaratumab,omalizumab, panitumumab, pembrolizumab, pertuzumab, ramucirumab,ranibizumab, raxibacumab, reslizumab, rinucumab, rituximab, sarilumab,secukinumab, siltuximab, tocilizumab, tocilizumab, trastuzumab,trevogrumab, ustekinumab, and vedolizumab.

In some embodiments, the protein of interest is a recombinant proteinthat contains an Fc moiety and another domain, (e.g., an Fc-fusionprotein). In some embodiments, an Fc-fusion protein is a receptorFc-fusion protein, which contains one or more extracellular domain(s) ofa receptor coupled to an Fc moiety. In some embodiments, the Fc moietycomprises a hinge region followed by a CH2 and CH3 domain of an IgG. Insome embodiments, the receptor Fc-fusion protein contains two or moredistinct receptor chains that bind to either a single ligand or multipleligands. For example, an Fc-fusion protein is a TRAP protein, such asfor example an IL-1 trap (e.g., rilonacept, which contains the IL-1RAcPligand binding region fused to the 11-1R1 extracellular region fused toFc of hIgG1; see U.S. Pat. No. 6,927,004, which is herein incorporatedby reference in its entirety), or a VEGF trap (e.g., aflibercept orziv-aflibercept, which comprises the Ig domain 2 of the VEGF receptorFlt1 fused to the Ig domain 3 of the VEGF receptor Flk1 fused to Fc ofhIgG1; see U.S. Pat. Nos. 7,087,411 and 7,279,159). In otherembodiments, an Fc-fusion protein is a ScFv-Fc-fusion protein, whichcontains one or more of one or more antigen-binding domain(s), such as avariable heavy chain fragment and a variable light chain fragment, of anantibody coupled to an Fc moiety.

C. Producing mAb with Native Disulfide Bond Pattern

One embodiment provides methods of producing a protein drug productcontaining less than 30% scrambled disulfide bonds. An exemplary methodincludes culturing cells producing the antibody in a cell culture undersuitable conditions to produce the antibody, purifying the antibodyunder suitable conditions to extract the antibody, admixing the antibodywith excipients under suitable conditions to stabilize the antibody,obtaining a sample of the antibody from the cell culture, followingpurification of the antibody from the cell culture, or following theaddition of excipients to the purified antibody, characterizingdisulfide bonds of the antibody according to the disclosed methods, andmodifying one or more cell culture, purification or excipient conditionsto reduce the amount of crossed hinge disulfide bonds of the antibody.

The one or more cell culture, purification, or excipient conditions thatare changed to reduce the amount of scrambled disulfide bonds in theantibody include but are not limited to temperature, pH, oxygen levels,reactive oxygen species, surfactants, or combinations thereof. In oneembodiment, an amino acid free strategy of cell culture could affectdisulfide bond formation.

In one embodiment, the cells producing the antibody are Chinese hamsterovary cells. In another embodiment, the cells are hybridoma cells.

In one embodiment, the protein drug product can have less than 30%scrambled disulfide bonds in the hinge region. The protein drug productcan have less than 30%, 25%, 20%, 18%, 16%, 14%, 12%, 10%, 9%, 8%, 7%,6%, 5%, 4%, 3%, 1%, 0.5%, or 0.1% scrambled disulfide bonds in the hingeregion.

In another embodiment, the protein drug product can have less than 10%scrambled disulfide bonds overall. The protein drug product can haveless than 10%, 9.5%, 9%, 8.5%, 8%, 7.5%, 7%, 6.5%, 6%, 5.5%, 5%, 4.5%,4%, 3.5%, 3%, 2.5%, 2%, 1.5%, 1%, 0.5%, or 0.1% scrambled disulfidebonds.

EXAMPLES Example 1: Synthesis of Parallel and Crossed Hinge Peptides

Methods:

Cross-Linking

Cysteine containing peptides were purchased from a commercial vendor.The peptides were cross-linked by incubation with 1 mM Cu²⁺ as theoxidant in the presence of air. The molar ratio of peptide to Cu²⁺ was5:1.

N-Terminal Analysis/Edman Degradation

The cross-linked peptides were suspended in water and were placed into aprotein sequencer. The peptides were exposed to phenyl isothiocyanate(PITC). PITC couples with the N-terminal residue to form a PTCpolypeptide. Trifluoroacetic acid was added to the reaction and the PTCN-terminal residue underwent acid cleavage, resulting in the release ofan unstable ATZ-amino acid. The ATZ-amino acid was separated from thepeptide solution into a conversion flask containing ethyl acetate. TheATZ-amino acid was converted into a stable PTH-amino acid with 25% TFA,v/v in water. The PTH-amino acid solution was injected onto an HPLC.Each amino acid of the peptide is identified by HPLC.

Results

Cu²⁺ has been reported to induce the formation of disulfide bonds byproducing radicals (Prudent, M., and Girault, H. H., Metallomics,1:157-165 (2009)). Peptides exposed to Cu²⁺ at a molar ratio ofpeptide/Cu²⁺ of 5/1 formed disulfide bonds as illustrated in FIG. 1A.The first oxidation formed a single, non-selective bond that was eitherparallel or crossed in nature (FIGS. 1A and 1C). During the seconddisulfide bond formation, parallel connectivity was found to be thepreferred connection (FIG. 1B). The peptide concentration was found toaffect the type of disulfide bond that was formed. A higherconcentration of peptide, 8 μg/ml, induced the formation of moreparallel hinge dimers than a concentration of 0.1 μg/ml (FIGS. 2A-2B).Higher peptide concentration favors inter-molecular bridges.

The identity of the peptides was confirmed using N-terminal analysis andEdman degradation (FIGS. 3A-3I, FIGS. 4A-4I, and FIGS. 5A-5B).

Example 2: Analysis of the Hinge Region of Two mAbs

Methods

Hinge DSB Characterization

Antibodies were first digested into peptides. IgG1 antibodies weresubjected to dual-enzyme digestion using proteinase K followed bytrypsin. IgG4 antibodies were subjected to digestion by FabRICATORfollowed by trypsin. Hinge peptide standards were prepared as above,using the hinge region sequence of the IgG1 and IgG4 antibodies toprepare the peptides. The digested peptide mixtures were subjected toLC-MS analysis. Hinge peptide standards were also subjected to LC-MAanalysis. Retention time analysis was performed to compare the retentiontime of the antibody peptides to the retention time of the hinge peptidestandards.

Results

IgG1 mAb1 was subjected to digestion into peptides and the resultingpeptides were subjected to LC/MS analysis. The hinge peptide standardsdescribed above were also subjected to LC/MS analysis. As shown in FIGS.6A-6C, IgG1 mAb1 has about 0.9% crossed hinge disulfide bonds.

A second antibody, IgG4 mAb1, was also analyzed using the disclosedmethods and hinge disulfide bond peptide standards. As shown in FIGS.7A-7C, IgG4 mAb1 had about 0.6% crossed hinge disulfide bonds.

While in the foregoing specification this invention has been describedin relation to certain embodiments thereof, and many details have beenput forth for the purpose of illustration, it will be apparent to thoseskilled in the art that the invention is susceptible to additionalembodiments and that certain of the details described herein can bevaried considerably without departing from the basic principles of theinvention.

All references cited herein are incorporated by reference in theirentirety. The present invention may be embodied in other specific formswithout departing from the spirit or essential attributes thereof and,accordingly, reference should be made to the appended claims, ratherthan to the foregoing specification, as indicating the scope of theinvention.

1-13. (canceled)
 14. A method of producing a protein drug product,comprising: producing the protein drug product in a cell culture;identifying scrambled disulfide bonds of the protein drug product; andmodifying one or more cell culture, purification or excipient conditionsto reduce the amount of crossed hinge disulfide bonds of the proteindrug product to less than 1.0%.
 15. The method of claim 14, wherein theone or more cell culture, purification or excipient conditions that aremodified are selected from the group consisting of temperature, pH,oxygen levels, reactive oxygen species, surfactants, or combinationsthereof.
 16. The method of claim 14, wherein the protein drug product isselected from the group consisting of an antibody, a fusion protein,recombinant protein, or a combination thereof.
 17. The method of claim14, wherein the identifying the scrambled disulfide bonds of the proteindrug product comprises: preparing peptide standards comprising regionsof the protein drug product containing one or more disulfide bonds. 18.The method of claim 17, wherein preparing the peptide standardscomprises using a peptide concentration of from 0.1 μg/mL to 0.8 μg/mL.19. The method of claim 17, wherein the disulfide bonds in the peptidestandards are formed through an oxidation reaction that comprisesexposing the peptide standard to zinc.
 20. The method of claim 17,wherein the disulfide bonds in the peptide standards are formed throughan oxidation reaction that comprises oxidation by air or chemical. 21.The method of claim 17, wherein the disulfide bonds in the peptidestandards are formed through an oxidation reaction that comprisesoxidation with Cu2+.
 22. The method of claim 21, wherein the molar ratioof peptide:Cu2+ for formation of disulfide bonds is 5:1.
 23. The methodof claim 17, wherein a first peptide standard comprises a firstscrambled disulfide bond, and a second standard comprises a second anddifferent scrambled disulfide bond.
 24. The method of claim 23, whereinthe first peptide standard and the second peptide standard havedifferent liquid chromatography retention times.
 25. The method of claim17, wherein identifying the scrambled disulfide bonds of the productdrug product further comprises: digesting a sample of the protein drugproduct into peptides; analyzing a sample containing the protein drugproduct peptides comprising one or more disulfide bonds and the peptidestandards comprising one or more disulfide bonds using an LC-MS² system;and comparing retention times of the sample containing protein drugproduct peptides comprising one or more disulfide bonds with theretention times of the peptide standards comprising one or moredisulfide bonds.
 26. The method of claim 25, wherein peptides detectedat the retention time of the first peptide standard indicates that thescrambled disulfide bonds of the first peptide standard are present inthe protein drug product, and peptides detected at the retention time ofthe second peptide standard indicates that the scrambled disulfide bondsof the second peptide standard are present in the protein drug product.27. The method of claim 25, wherein digesting the sample comprisestryptic digestion or dual-enzyme digestion.
 28. The method of claim 14,wherein the scrambled disulfide bonds are selected from the groupconsisting of crossed disulfide bonds, crisscrossed disulfide bonds, andintra-chain disulfide bonds.
 29. The method of claim 17, wherein the oneor more disulfide bonds are in the hinge region of an antibody.
 30. Themethod of claim 17, wherein the method further comprises: quantifyingthe amount of one or more disulfide bonds present in the protein drugproduct.
 31. The method of claim 16, wherein the protein drug product isa fusion protein.
 32. The method of claim 31, wherein the fusion proteinis an Fc fusion protein.
 33. The method of claim 14, wherein the proteindrug product has less than 30% scrambled disulfide bonds.